This program explores innate anti-microbial defense, pro-inflammatory and other signaling mechanisms involving deliberate reactive oxygen species (ROS) production by Nox family NADPH oxidases. Our current focus investigates cellular mechanisms regulating nonphagocytic Nox family NADPH oxidases expressed primarily in epithelial cells (Nox1, Nox4, Duox1, Duox2). Tissues studied include mucosal surfaces (lung and gastrointestinal tract), liver, kidney, thyroid and exocrine glands (salivary, mammary), and vascular tissues. ROS produced by these oxidases affect responses to infection, growth factors, hormones, cytokines, cell differentiation, cellular senescence, programmed cell death (apoptosis) and oxygen sensing. Several non-phagocytic Nox enzymes also serve in host defense and inflammatory processes, as they are expressed predominately on apical surfaces of epithelial cells and are induced or activated by cytokines or by recognition of pathogen-associated molecular patterns. Recently, we found Nox4 is induced in Hepatitis C virus (HCV)-infected hepatocytes and is a source of excess chronic ROS (oxidative stress) that can lead to liver fibrosis (cirrhosis). The induction of Nox4 by HCV involves autocrine TGF-beta stimulation of Nox4 expression, consistent with its induction by TGF-beta in several tissues susceptible to fibrotic disease (lung, kidney, liver, heart). In 2012, we studied Nox4 induction by TGF-beta in the context of the epithelial-to-mesenchymal transition (EMT), a process in which cells assume increased plasticity during normal development and wound healing that also functions in pathological settings such as fibrosis and tumor metastasis. Our studies show that Nox4 serves a redox-based signaling role promoting the EMT and cell migration in normal and metastatic breast epithelial cells. We found TGF-beta induces Nox4 (mRNA and protein) and ROS generation in normal (MCF10A) and metastatic (MDA-MB-231) human breast epithelial cells, whereas cells expressing dominant-negative (DN) Nox4 or Nox4-targeted shRNA show lower ROS production in response to TGF-beta. Constitutively active TGF-beta receptor type I significantly increased Nox4 promoter activity, mRNA and protein, and ROS generation. Nox4 transcriptional regulation by TGF-beta was SMAD3-dependent, based on effects of constitutively active SMAD3, whereas DN SMAD3 or SIS3, a SMAD3 inhibitor, had the opposite effects. Nox4 knockdown, DN Nox4 or SMAD3, or SIS3 also blunted TGF-beta induced wound healing and cell migration. Finally, we showed that Nox4 plays a role in TGF-beta regulated fibronectin expression, based on the effects of DN Nox4 in reducing fibronectin mRNA in TGF-beta treated cells. These data indicate Nox4 contributes to NADPH oxidase-dependent ROS production critical for EMT progression and migration of breast epithelial cells. These observations suggest Nox4 as a potential target for therapeutic intervention to affect wound healing, fibrosis, cancer progression and metastasis. Our studies on Duox-reconstituted cell models have been used to identify several Duox single nucleotide polymorphisms (SNPs) and mutations that alter oxidase function or cellular targeting, six of which have been linked to congenital hypothyroidism. Other common polymorphisms exhibiting altered Duox activity are being screened in patient populations for links to altered susceptibilities to infectious or inflammatory disease (asthma, bacterial or viral infection, cystic fibrosis). We have also undertaken a survey of murine strain-specific Duox polymorphisms that may predispose mice to colitis in the absence of glutathione peroxidase 1 and 2. Related experiments are exploring effects of microbial agonists that activate Duox2 in gut epithelial cells that would thereby stimulate inflammatory responses. Circulating phagocytes generate high levels of ROS that serve as important microbicidal agents in response to infectious or inflammatory stimuli, which is attributed to NADPH oxidase activation (Nox2- or phox-based enzyme). Patients with chronic granulomatous disease (CGD) suffer from NADPH oxidase deficiencies resulting in enhanced susceptibility to microbial infections and aberrant inflammatory responses. In collaborative studies, we are examining roles of pro-oxidants as direct activators the phagocytic NADPH oxidase. We showed that membrane translocation of p40phox can be triggered by hydrogen peroxide or arachidonic acid, leading to assembly and activation of other oxidase components. Other studies showed that the antioxidant enzyme, peroxiredoxin 6, associates and co-migrates with cytosolic phox proteins and serves as a positive regulator of oxidase activity. Its oxidase-supportive function was shown to involve phospholipase A2 activity that releases arachidonate. Finally, the redox-active Pseudomonas aeruginosa toxin, pyocyanin, was also shown to trigger oxidase activation through generation of intracellular oxidants. These studies demonstrating that pro-oxidants can act in a positive feed-forward signaling mechanism to promote excess ROS generation provide insight on mechanisms of pro-inflammatory lung disease development occurring in cases of chronic Pseudomonas infection (i.e., cystic fibrosis).